The Clinical Significance Of The Molecular Genetic And Pathologic Heterogeneity In The Intermediate Stage Hepatocellnlar Carcinoma

Hepatocellular carcinoma is one of the most common fatal malignant tumors of human beings,with an obvious intratumor heterogeneity and a poor prognosis.Intermediate stage hepatocellular carcinoma is commonly occurred at disease diagnosis,with varied clinicopathological characteristics,therapeutic options and patient's survival outcomes.Intratumor heterogeneity can be found in many solid tumors,resulting in complexity of tumor characteristics in terms of space and time,and then affects the patient's tumor classification,treatment effeciency,drug resistance and prognosis.Recently,the concept of "Liquid Biopsy" was proposed,mainly containing the circulating tumor DNA and circulating tumor cells techniques.Liquid biopsy technique can accomplish the sequencing of the blood DNA samples with high depth and high precision,reflecting more comprehensive mutation characteristics of the overall tumor variation than traditional biopsy.In view of huge heterogeneity of intermediate HCC in tumor characteristics,treatment and prognosis,we design studies proceeding from the following two aspects:PART ONE: The feasibility study of using ctDNA-NGS technique to evaluate the intraheterogeneity of the intermediate stage of hepatocellular carcinoma.Objective: This study aimed to evaluate the genetic intratumor heterogeneity of the intermediate HCC patients and explore the application potential of liquid biopsy in tumor heterogeneity,drug resistance screening and prognosis prediction.Methods: Specific regions based capture sequencing was used for DNA samples from the multiple point tissues,plasma and WBC of five patients with intermediate stage HCC.Bioinformatics analysis was used to analysis the distribution of somatic mutation in different samples.Then the pathway enrichment analysis was used to reveal key signaling pathways which related to the mutated genes.Results: Two CTCs in each of three patients and one CTC in one patient were detected in five patients with intermediate stage HCC,and mean sequencing depth of 5 cfDNA samples is 5711×,while median mutation allele frequency(MAF)of all somatic mutation in cfDNA is 28.34%(interquartile range: 6.89%-39.49%).25 somatic variants were detected in 4 patients,and the number of each are 3,4,6 and 12.For the four patients,6 in all 12(50%)tissue samples can't cover all mutations which detected in one's patients,however,100% of tissue related mutations can be validated in cfDNA samples.Conclusions: Liquid biopsy technique,especially the circulating tumor DNA as the sequencing samples,can better reflect the overall genetic characteristics of the tumor than traditional biopsy method.PART TWO: Prognostic efficiency of the substaging model for patients who underwent hepatectomy for intermediate stage hepatocellular carcinomaObjective: This study aimed to validate the predictive performance of the Bolondi's substaging model for postoperative survival outcomes in patients who underwent hepatectomy for hepatocellular carcinoma(HCC)at BCLC intermediate stage.Methods: Between January 2008 and February 2010,a total of 343 patients with intermediate stage HCC who underwent hepatectomy were enrolled.Patients were stratified into 4 substages(B1–B4)based on the Bolondi's substaging model.The last follow-up was conducted at February 2014.The endpoint of this study was overall survival(OS).The OS of patients at different substages were compared using the Kaplan-Meier method and the Log-rank test.The independent risk factors associated with OS was analyzed using the Cox hazards model.Results: Of these patients,143,183,12 and 5 patients were classified as the substages of B1,B2,B3,and B4,respectively.Their 5-year OS rate was 45.5%,30.4%,13.0%,and 0%,and the median survival was 55.1,35.1,14.4 and 4.7 months,respectively(P < 0.001).On Cox multivariable analysis,preoperative serum albumin level ? 35g/L(hazard ratio: 2.82,95% confidence interval: 1.37-5.8),hemoglobin level ? low limit of normal(2.16,1.27-3.96),surgical margin invasion(2.93,1.33-6.44),presence of microvascular invasion(1.53,1.06-2.22),and tumor number > 5(1.48,1.05-2.09)were the independent risk factors of OS in the B1 and B2 substage patients.Conclusions: The substaging model produced an optimal OS prediction for HCC patients at BCLC intermediate stage after hepatectomy.The B1 and B2 substage patients achieved better long-term survival outcomes after surgery.